An Introduction to Monoclonal Antibodies and Their Uses

The way problems are approached in the medical field is changing thanks to new discoveries and technological advancements. A key discovery that drove such dramatic change was the discovery of monoclonal antibodies (mAb). From diagnosing diseases to helping cure cancer, monoclonal antibodies have paved the way to an advanced time in science.

An Introduction to Monoclonal Antibodies

Since their discovery in 1975, these man-made proteins - which act like human antibodies found in the immune system - have provoked a new era of research and discovery [1, 2]. They are created to target specific antigens and carry out a desired function on the chosen cells. Before we delve into the vast world of monoclonal antibodies, it is important to have a clear understanding of what antibodies are and how they work in our immune system. Also referred to as immunoglobulins, these protective proteins produced by B lymphocytes respond to the presence of a foreign substance by attaching to its antigens, ultimately killing or neutralising it [3]. 

Their unique property of being able to identify specific cells in the body has encouraged scientists to examine their potential uses. A result of this was the extraordinary discovery made by two immunologists, George Kohler and Cesar Milstein, who won a Nobel Prize for their work. They developed a way to produce these new antibodies by fusing myeloma cells (a type of cancerous immune cell) with mouse spleen cells that had been exposed to a specific target [4].  Since then, monoclonal antibodies have significantly shaped the medical world. 

There are four main types of antibodies: murine, which are made from mouse proteins; chimeric, which are made from a combination of mouse and human proteins; humanised, which are made from small parts of mouse proteins attached to human proteins; and finally, human, which are fully human antibodies [1]. It is exceptionally clear that monoclonal antibodies have greatly benefited modern medicine. However, their wide range of side effects raises several questions.

The Production of Monoclonal Antibodies

When making monoclonal antibodies, an animal (usually a mouse) is exposed to a specific target (usually a pathogen). The production of B lymphocytes is encouraged as a resulting immune response to the foreign substance (the target/pathogen). The lymphocytes are then collected from the spleen or lymph nodes. Since these cells do not have a high survival rate when cultured, they are fused with myeloma cells (cancerous B cells) to form a hybridoma. This fusion can be done using a substance called polyethylene glycol (PEG) or by electric pulses. Both processes disrupt the cell membranes, allowing the cells to fuse. This fusion, however, is not 100% effective. So, to separate unwanted substances, the products are placed in a selective medium – a hypoxanthine-aminopterin-thymidine (HAT) medium which inhibits DNA synthesis. B cells and hybridomas can overcome this as they produce thymidine kinase, which allows the synthesis of DNA polymerase (an enzyme used in DNA synthesis) from thymidine supplied by the medium. Non-fused myeloma cells do not produce thymidine kinase and so cannot undergo DNA synthesis nor survive. Due to their short survival rate, B cells also eventually die off, with only hybridomas remaining. These are then screened for the desired antibodies and are grown in cultures to then be extracted and purified for further use [5].

Use in Diagnostics

The use of monoclonal antibodies has revolutionised diagnostics due to their ability to quickly and easily identify various diseases. A popular use of monoclonal antibodies is in immunoassays - rapid, accurate tests that can be used to detect specific molecules by using antigens or antibodies. Monoclonal-polyclonal immunoassays are used to detect antigens. The monoclonal antibody is absorbed onto a plastic microtiter plate, and as the sample is added to the plate, the antibodies bind to the target antigens and retain it. A polyclonal antibody is then added, which also binds to the antigen. This forms a monoclonal-polyclonal sandwich, as the antigen is sandwiched between the monoclonal and polyclonal antibodies. A colourimetric substance is then added or may have already been attached to the antibodies. The substrate will then detect the sandwiches and generate a colour proportional to the number of antigens present in the sample, so a quantitative result can be produced by analysing the microtiter plates on the colourimeter [6]. A great advantage of this is that it is easy to measure the amount of the specified foreign substance in the sample and give treatment accordingly. Examples of diseases that can be identified by immunoassays include malaria and chlamydia. However, there are some downsides to this method, such as the fact that it can only be done in a laboratory.  This means it cannot be used during an urgent crisis.

Organ Rejection

Another key use of this extraordinary protein is the prevention of organ rejection and allowing organ transplantation. The antibodies are used for this work in a variety of ways, but they all share a common feature of targeting the cluster of differentiation (CD) proteins - molecules that help differentiate one cell type from another - found on the surfaces of T and B cells [7]. Examples of antibodies used to suppress organ rejection include monoclonal antibodies against the proteins CD3, CD25 and CD52, which are found on immune cells and are usually involved in controlling proliferation [8]. The OKT3 antibodies act against the CD3 proteins on the receptors found on the T cells. They were used in the setting of renal transplants in the early 1980s, however due to large number of side effects, they have now been removed from the market. After the antibody binds, T cells are no longer able to proliferate or differentiate and so are no longer seen in circulation. Due to the low quantities of T cells in the blood, the immune system does not react as effectively to the transplanted organ, reducing organ rejection.

Many other drugs and methods are now being optimised and used due to the large number of side effects this antibody could cause. The first dose itself may cause a few complications including short-term physiological changes which resemble systemic inflammatory response syndrome. These could be seen as a high fever, hypotension, chills, nausea, vomiting, diarrhoea, dyspnea and even pulmonary oedema (excess fluid in the lungs). In rare cases, the use of this antibody could also result in aseptic meningitis. In addition to these complications, since these antibodies are from mice, they also induce the development of anti-murine antibodies (human anti-mouse antibodies), limiting their effectiveness. Scientists have now learnt from these drawbacks and have created new antibodies that inhibit the proteins CD25 AND CD52 instead [8]. Being quite a recent discovery, monoclonal antibodies still have some room to reach perfection, but solutions from dilemmas such as these pave a way for a bright future for these fascinating proteins.

Cancer

One of the most iconic uses of these antibodies includes their involvement in treating cancer, where they can be used as a type of targeted therapy. An example of their use includes their prevailing presence in immunotherapy, as they assist the immune system to act against the cancerous cells. A key antibody used is rituximab, which binds to a protein called CD20 found on B cells; this protein encourages the immune system to detect any B cells with the antibody bound to them and kill them [9]. As a result, unwanted B cells are killed and cancers like leukaemia and non-Hodgkin lymphoma can be treated far more effectively. Another way monoclonal antibodies can be used is by bringing T cells closer to cancers, to help the immune system to kill them. These are bispecific antibodies – meaning they act on two different proteins. A key example includes blinatumomab (used to treat acute lymphoblastic leukaemia), which binds to the CD19 protein found on cancerous B cells and the CD3 protein found on the T cells. This helps the T cells get close enough to the cancer cells to respond and kill them [9]. 

Other uses of monoclonal antibodies to treat cancer include attacking or blocking antigens on cancerous cells or nearby cells that help it grow or spread. An example includes the antibody trastuzumab, which acts against the HER2 proteins that are usually found in large numbers on breast and stomach cancer cells [1]. When activated, HER2 proteins help the cancerous cells grow. By binding to these proteins, they are stopped from being activated and can no longer help the growth of cancer.  Trastuzumab is an example of a naked mAb, which work by themselves and are the most common type of antibody used to treat cancer. There are also conjugated monoclonal antibodies which are combined with a chemotherapy drug or a radioactive particle. The antibodies find and attack the targeted antigens and deliver the toxic substance or radiation where it is needed most [1].

Even though monoclonal antibodies provide huge advantages and opportunities to treat a variety of cancers, they have their flaws. Since antibodies are proteins, they can cause allergic reactions, inducing fever, weakness, headache, nausea, vomiting, diarrhoea, low blood pressure and rashes. Targeted antibodies can also cause specific symptoms. An example includes bevacizumab, which targets a protein called VEGF that affects tumour blood vessel growth [1], and is used to treat advanced cancers of the lung, colon and rectum [10]. The side effects it may cause include high blood pressure, bleeding, poor wound healing, blood clots and kidney damage. The vast range of side effects usually impacts how often these antibodies are used, limiting their potential [1]. Before treatment with monoclonal antibodies, a specialist needs a sample (biopsy) of the cancer, to find out if the treatment will be effective by looking for specific proteins and genes. The conditions that must be met for monoclonal antibodies are highly specific, so this treatment may not be available to everyone, yet again limiting its use. However, despite these drawbacks, due to their specificity, when used effectively, they can cause a significant reduction in cancer cells and limit the damage caused on nearby cells.

COVID-19

As the use of monoclonal antibodies increases and the efficacy is enhanced, these proteins are helping to provide solutions against the COVID-19 virus. This can ultimately help achieve SDG 3: Good Health and Well-being, as there could be a reduced COVID-19 infection rate with these solutions. They work by binding to the trimeric spike (S) glycoprotein found on the COVID-19 viral surface. The virus uses this spike to mediate entry into host cells, so by preventing the S protein from functioning, the virus can no longer enter our cells or affect us. Antibodies that are used to treat COVID-19 include casirivimab and imdevimab. Their effectiveness was seen through the double-blind, placebo-controlled clinical trial that took place with 799 non-hospitalised adults with mild to moderate COVID-19 symptoms. At day seven, the reduction in the quantity of viruses in patients treated with the antibody was larger than in patients treated with a placebo. A key indication of the effectiveness of the antibodies is that hospitalisations and emergency room visits within 28 days after treatment occurred in 3% of monoclonal antibody-treated patients on average, compared to 9% in placebo-treated patients [11]. However, despite their evident advantages, the antibodies were associated with worse clinical outcomes when administered to hospitalised patients requiring high-flow oxygen or mechanical ventilation. Possible side effects of this treatment include anaphylaxis and infusion-related reactions, fever, chills, hives, and itches. Despite these negatives, the benefits these proteins provide outweigh the possible drawbacks as they can be useful in preventing hospitalisation [11]. Specific monoclonal antibodies, however, have recently been found to be less effective in treating different variants, especially those with   L452R or E484K substitution in the spike protein, as it is seen to cause reduced susceptibility and sensitivity to monoclonal antibodies that are being used to treat COVID-19, such as bamlanivimab, etesevimab and casirivimab [12].

What the Future Holds

Since their discovery in 1975, the use of monoclonal antibodies has grown rapidly and has paved a way for drastic changes in many aspects of the medical world. From diagnostics to cancer treatment, these antibodies have found quick and effective solutions for a variety of problems. Despite their many side effects, their future remains bright as scientists constantly learn from the setbacks and discover more about these fascinating proteins. Due to their versatile nature, they can be used in oncology, neurology, immunology, metabolic and cardiovascular diseases. In the past three decades, about 50 monoclonal antibodies have been approved, and in 2017, 5 of the top 10 selling drugs in the global market were monoclonal antibodies [13]. This itself highlights the great potential and future these therapeutic proteins hold. The constantly evolving use of monoclonal antibodies has made them one of the most useful innovations in today’s medical society and is expected that many more of their wonders will be revealed in the coming years [13].

References

[1] The American Cancer Society medical and editorial content team, “Monoclonal Antibodies and Their Side Effects”, American Cancer Society, December 27, 2019. [Online]. Available: https://www.cancer.org/treatment/treatments-and-side-effects/treatment-types/immunotherapy/monoclonal-antibodies.html. [Accessed 3 January 2021].

[2] The Editors of Encyclopaedia Britannica, “Monoclonal antibody,” Britannica, October 12, 2018. [Online]. Available: https://www.britannica.com/science/monoclonal-antibody.  [Accessed 10 January 2021].

[3] The Editors of Encyclopaedia Britannica, “Antibody,” Britannica, May 27, 2020. [Online]. Available: https://www.britannica.com/science/antibody. [Accessed 10 January 2021].

[4] Guest Author, “The story of a revolution: César Milstein and monoclonal antibodies,” Medical Research Council, February 14, 2013. [Online]. Available: https://mrc.ukri.org/news/blog/the-story-of-a-revolution-cesar-milstein-and-monoclonal-antibodies/?redirected-from-wordpress. [Accessed 30 December 2020].

[5] Anon., “Monoclonal Antibodies Overview”, GenScript, n.d. [Online]. Available: https://www.genscript.com/how-to-make-monoclonal-antibodies.html. [Accessed 30 December 2020].

[6] Anon., “Back to Basics: What is an immunoassay?”, ImmunoChemistry Technologies, July, 2016. [Online]. Available: https://immunochemistry.com/2016/07/07/back-basics-immunoassay/#:~:text=Immunoassays%20rely%20on%20the%20inherent,specific%20structure%20of%20a%20molecule.&text=Once%20purified%20from%20the%20blood,limited%20interferences%20from%20other%20substances. [Accessed 3 January 2021].

[7] I. Mallik, “CD Markers in Cancer Diagnosis and Treatment”, verywellhealth, January 20, 2020. [Online]. Available: https://www.verywellhealth.com/understanding-cd-markers-2252468. [Accessed 20 May 2021].

[8] N. Mahmud, D. Klipa and N. Ahsan, “Antibody immunosuppressive therapy in solid-organ transplant,” Taylor & Francis Online, vol. 2, issue 2, pp. 148-156, 2010. Available: https://doi.org/10.4161/mabs.2.2.11159.

[9] Anon., “Monoclonal Antibodies,” National Cancer Institute, September 24, 2019. [Online]. Available: https://www.cancer.gov/about-cancer/treatment/types/immunotherapy/monoclonal-antibodies#:~:text=For%20example%2C%20some%20monoclonal%20antibodies,immune%20system%20to%20kill%20them. [Access 3 January 2021].

[10] Anon., “Avastin”, Breastcancer.org, April 27, 2020. [Online]. Available: https://www.breastcancer.org/treatment/targeted_therapies/avastin#:~:text=Avastin%20is%20also%20approved%20by,will%20not%20affect%20those%20approvals. [Accessed 20 May 2020].

[11] Anon., “Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal Antibodies for Treatment of COVID-19,” U.S. Food and Drug Administration, November 21, 2020. [Online]. Available: https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-monoclonal-antibodies-treatment-covid-19#:~:text=Monoclonal%20antibodies%2C%20such%20as%20casirivimab,health%20during%20this%20unprecedented%20pandemic. [Accessed 4 January 2021].

[12] Anon., “SARS-CoV-2 Variant Classifications and Definitions”, Centers for Disease Control and Prevention, June 10, 2021. [Online]. Available: https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html. [Accessed 20 May 2021].

[13] B. J. Mills, E. M. Moussa, F. Jameel, “Chapter 1: Monoclonal Antibodies: Structure, Physicochemical Stability, and Protein Engineering”, Springer Link, March 14, 2020. [Online]. Available: https://link.springer.com/chapter/10.1007/978-3-030-31415-6_1. [Accessed 4 January 2021].